The long range objective of the proposed study is to define clearly the properties of mitochondrially bound forms of hexokinase in tumor cells, and to establish both roles of these enzymatic forms in the regulation of glucose utilization by neoplastic tissues, and their requirement for tumor cell growth. Preliminary work on this project has a) shown that all highly glycolytic tumors examined to date have mitochondrially associated hexokinase (J. Biol. Chem. 256, 8699, 1981); b) shown that the mitochondrially associated form of the enzyme is responsible, at least in part, for the high glucose utilization rate of rapidly growing cancer cells (PNAS 74, 3735, 1977; J. Biol. Chem. 256, 8699, 1981); c) defined conditions for solubilizing much of the hexokinase activity associated with hepatoma mitochondria (Biochem. 19, 4972, 1980); and d) identified at least two chromatographically distinguishable forms of the enzyme associated with tumor mitochondria (see preliminary results). Future experiments will focus on 6 specific ais: I) Conditions will be worked out to solubilize all the hexokinase activity associated with tumor mitochondria and to identify the total number of enzymatic species. II) Submitochondrial localization studies will be carried out to establish whether different enzymatic species have similar or different localizations. III) Mitochondrial forms of the enzyme will be purified from tumors, characterized, and tested for their ability to rebind to tumor and normal mitochondria. IV) Puriifed forms of mitochondrial hexokinase will be enzymatically and chemically modified prior to rebinding in order to odemtofu specific amino acid residues involved in binding. V) Mitochondrial receptors for hexokinase in tumor mitochondria will be solubilized, and attempts will be made to purify and characterize them. VI) Lonidamine, which inhibits the mitochondrial form of hexokinase in Ehrlich ascites cells, will be tested both for its ability to inhibit the mitochondrial form of the enzyme in other tumors, and its ability to suppress tumor cell growth in tissue culture and in tumor bearing animals. The proposed studies are both necessary and fundamental to our understanding of the most common biochemical phenotype of cancer cells, namely their propensity to catabolize large amounts of glucose relative to many normal cells. These studies may also provde useful information about the efficacy of inhibitors of glucose catabolism as inhibitors of tumor cell growth.